Full Report on

Exercise 4

ENZYME KINETICS

John Patricia Mae Centeno

CHEM 161.1-3L

2nd Semester AY 2018-2019

Groupmates:
Janelle Allyza Conti

Earlene Lagasca

Jose Lorenzo Manansala

Date performed: 01 March 2019

Date Submitted: 08 March 2019

Aldwin Ralph Briones, RCh.

Laboratory Instructor
 I. Results and Discussion

Enzymes are considered to be the catalysts of biological system; central to every

biochemical process. It acts in organized sequences, catalyzing hundreds of stepwise reactions
that degrade nutrient molecules, conserve and transform chemical energy, and make biological
macromolecules from simple precursors. Almost all enzymes are proteins except from a small
group of catalytic RNA molecules. Their catalytic activity depends on the integrity of their native
protein conformation. If an enzyme is denatured or dissociated into its subunits, catalytic activity
is usually lost. If an enzyme is broken down into its component amino acids, its catalytic activity
is always destroyed (Nelson and Cox, 2017).

The study of enzymes has vast practical importance. In some diseases, especially
inheritable genetic disorders, there may be a deficiency or even a total absence of one or more
enzymes. For other disease conditions, excessive activity of an enzyme may be the cause.
Measurements of the activities of enzymes in blood plasma, erythrocytes, or tissue samples are
important in diagnosing certain illnesses (Nelson and Cox, 2017).

In this experiment, invertase is the enzyme of interest. Invertase, also called beta-
fructofuranosidase, which cleavs the terminal non-reducing end of betafructofuranoside residues,
is a glycoprotein with an optimum pH 4.5 and stability at 50 C. Invertase in nature exists in
different isoforms. In yeasts, it is present either as extracellular Invertase or intracellular
Invertase. In plants, there are three isoforms each differing in biochemical properties and
subcellular locations. Invertase in plants is essential not only for metabolism but also help in
osmoregulation, development and defence system. In humans, the enzyme acts as an immune
booster, as an antioxidant, an antiseptic and helpful for bone cancer or stomach cancer patients
in some cases. Invertase is an enzyme that catalyzes the breakdown of sucrose to glucose and
fructose (Kulshrestha et al., 2013). The amount of sucrose consumed or the amount of reducing
sugars formed are correlated with the enzymatic activity of invertase. However, the amount of
sucrose consumed is difficult to determine. Thus, the amount of glucose and fructose produced
are the subject of interest to correlate it with the enzyme activity. The method used in determining
the amount of reducing sugars is the Nelson Method.

In this experiment, the Nelson’s method was utilized for the analysis of reducing sugars,
the effect of incubation time on product formation, substrate concentration, addition of an
inhibitor to enzyme catalysis, and varying pH and temperature were determined.
 In the first part of the experiment, a standard curve was constructed in order to determine
if the relationship between the glucose concentration and the absorbance for the standard is
linear enough to be utilized as a standard. Initially, 10 test tubes of 2 mM of different glucose
concentrations were used. Distilled water and Nelson’s reagent. After that, the tubes were
covered with marble placing it in a boiling water bath for 20 minutes. After that, the solutions
were then cooled to room temperature before 1.00 mL of arsenomolybdate reagent was added
to each tube. The contents of each tube were mixed using a vortex stirrer, and was allowed to
stand for 5 minutes at room temperature. Then, 7.00 mL of distilled water, and was re-mixed
using the vortex mixer. The absorbance of each solution was determined at 510 nm wavelength.
The data are shown in table 3.1.

Table 3.1. Data on the construction of the standard curve.

Sample Glucose Absorbance

No. concentration

(mM)

1 0 0

2 0.1 0.1027

3 0.2 0.1498

4 0.4 0.3146

5 0.8 0.6334

6 1.2 0.9147

7 1.6 1.1998

8 2 1.5085

R2 0.9995

m 0.748 AU/mM

b 0.0139 AU

Figure 1.1. The standard curve for the determination of the reducing sugar.

Standard Curve
1.6
1.4 y = 0.748x + 0.0139
R² = 0.9995
1.2
ce

1
 II. Sample Calculations

Preparation of glucose solution for standard

𝐶1 𝑉1 = 𝐶2 𝑉2

(5.0 𝑚𝐿)(20 𝑚𝑀) = (𝑉2 )(2 𝑚𝑀)

𝑉2 = 50 𝑚𝐿

Concentration of Reducing Sugar

𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 − (𝑦 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡)
𝜇𝑚𝑜𝑙 𝑟𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑠𝑢𝑔𝑎𝑟 =
𝑠𝑙𝑜𝑝𝑒

0.0992 − 0.01392404
=
0.747953091
𝜇𝑚𝑜𝑙
= 0.114012179 .
𝑚𝐿

Enzyme Activity
 𝜇𝑚𝑜𝑙 𝑟𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑠𝑢𝑔𝑎𝑟
𝐸𝑛𝑧𝑦𝑚𝑒 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 =
𝑡𝑖𝑚𝑒 𝑒𝑙𝑎𝑝𝑠𝑒𝑑 (𝑚𝑖𝑛)

𝜇𝑚𝑜𝑙
0.114012 𝑚𝐿
=
5 𝑚𝑖𝑛.

= 0.022802 𝑈

Volumetric Activity

𝐸𝑛𝑧𝑦𝑚𝑒 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦
𝑉𝑜𝑙𝑢𝑚𝑒𝑡𝑟𝑖𝑐 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 =
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑢𝑡𝑖𝑙𝑧𝑖𝑒𝑑 (𝑚𝐿)
0.022802 𝑈
= 0.1 𝑚𝐿

= 0.22802 𝑈/𝑚𝐿

Initial Velocity

𝜇𝑚𝑜𝑙 𝑟𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑠𝑢𝑔𝑎𝑟

𝜈𝑜 =
𝑡𝑖𝑚𝑒 𝑒𝑙𝑎𝑝𝑠𝑒𝑑 (𝑚𝑖𝑛. )
0.076309
= 5

𝑚𝑀
= 0.01526185
𝑚𝑖𝑛.

Vmax

1
𝑉𝑚𝑎𝑥 =
𝑦 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡 𝑜𝑓 𝑡ℎ𝑒 𝐿𝐵 𝑝𝑙𝑜𝑡

1
=
1.212881503
𝑚𝑀
= 0.824482852 .
𝑚𝑖𝑛

Michaelis-Menten Constant

𝐾𝑚 = (𝑠𝑙𝑜𝑝𝑒)( 𝑉𝑚𝑎𝑥)

= (163.8462296)(0.824482852)

= 135.088406
 III. Reference/ Literature Cited

Boyer, R. F. (2011). Biochemistry laboratory: Modern theory and techniques (2nd ed.). Boston,
MA: Prentice Hall.

Kulshrestha, S., Tyagi, P., Sindhi, V., & Yadavilli, K. S. (2013). Invertase and its applications – A
brief review. Journal of Pharmacy Research,7(9), 792-797.
doi:10.1016/j.jopr.2013.07.014

Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2017). Lehninger Principles of biochemistry.
Macmillan Higher Education: Basingstoke.

Voet, J., & Voet, D. (2011). Biochemistry (4th ed.). New York: John Wiley & Sons, Inc.